CN115414486A - Dual-effect inhibitor of VEGFA signal and FGF-2 signal, new application of FGF-2 signal inhibitor - Google Patents

Abstract

The application provides a VEGFA signal, an FGF-2 signal double-effect inhibitor and new application of the FGF-2 signal inhibitor. Use of a VEGFA signalling and a dual effect inhibitor of FGF-2 signalling and/or an inhibitor of FGF-2 signalling for the preparation of a medicament for the treatment of tumours which are resistant to inhibitors of VEGFA signalling. The medicine can inhibit drug resistance of tumor to VEGFA signal inhibitor, and improve therapeutic effect on tumor.

Description

New dual-effect inhibitors of VEGFA signaling and FGF-2 signaling, FGF-2 signaling inhibitors use

technical field

The present application relates to the field of pharmaceutical technology, in particular to the novel use of dual-effect inhibitors of VEGFA signaling and FGF-2 signaling, and FGF-2 signaling inhibitors.

Background technique

Nasopharyngeal carcinoma is a malignant epithelial tumor that can be divided into keratinizing squamous cell carcinoma, nonkeratinizing squamous cell carcinoma, and undifferentiated or poorly differentiated carcinoma. Because nasopharyngeal carcinoma occurs in a special anatomical location, radiotherapy is currently the main method for the treatment of non-metastatic nasopharyngeal carcinoma. In recent years, the combination of adjuvant chemotherapy and radiotherapy and chemotherapy can significantly prolong the overall survival rate of patients. For other malignant solid tumors, anti-angiogenic drugs such as VEGFA signaling inhibitors are often used clinically to treat patients, which can effectively improve the survival of patients. However, after clinical trials, it was found that VEGFA signaling inhibitors, such as bevacizumab, are not effective in the treatment of nasopharyngeal carcinoma, which is probably due to the resistance of nasopharyngeal carcinoma to VEGFA signaling inhibitors.

Therefore, it is necessary to provide a method for treating nasopharyngeal carcinoma resistant to VEGFA signaling inhibitors, and to provide a method for improving drug resistance of nasopharyngeal carcinoma to VEGFA signaling inhibitors, thereby increasing the drug selection of nasopharyngeal carcinoma patients , to improve patient survival.

Tumor angiogenesis is very important for tumor growth and metastasis. The main signaling molecules that mediate angiogenesis include VEGFA, PDGF, FGF and so on. They activate downstream signaling pathways and promote angiogenesis by acting on the corresponding receptors of endothelial cells in tumors. VEGFA signal inhibitors can block VEGFA ligands and/or receptors and/or downstream signals, inhibit angiogenesis, effectively block the supply of oxygen and nutrients in tumors, and achieve the purpose of treating tumors. However, in some cases, tumors compensatoryly maintain angiogenesis by expressing other types of angiogenic signals, resulting in resistance to VEGFA signaling inhibitors.

Contents of the invention

In view of this, the application provides the application of VEGFA signaling and FGF-2 signaling dual-effect inhibitors and/or FGF-2 signaling inhibitors in the preparation of drugs for treating tumors, and the use of FGF-2 signaling inhibitors in the preparation of improving tumor response to VEGFA Drug application in signaling inhibitor resistance.

In a first aspect, the present application provides a dual-effect inhibitor of VEGFA signaling and FGF-2 signaling and/or an application of an FGF-2 signaling inhibitor in the preparation of a drug for treating tumors that are resistant to VEGFA signaling inhibitors .

Optionally, the dual-effect inhibitor of VEGFA signaling and FGF-2 signaling can simultaneously inhibit the VEGFA signaling pathway and the FGF signaling pathway of endothelial cells in the tumor, thereby overcoming the drug resistance of the tumor to the VEGFA signaling inhibitor, Inhibit tumor angiogenesis.

Optionally, the dual-effect inhibitor of VEGFA signaling and FGF-2 signaling includes lenvatinib.

Optionally, the FGF-2 signaling inhibitor inhibits at least one of the FGF-2 ligand, FGFR1 receptor and its downstream signaling pathway key molecule MYC, and the FGF-2 signaling inhibitor includes chemical drugs, polypeptides At least one of drugs, protein drugs and gene drugs.

Optionally, the VEGFA signaling inhibitor inhibits at least one of the VEGFA ligand, VEGFR2 receptor and its downstream signaling pathway key molecule MYC, and the VEGFA signaling inhibitor includes chemical drugs, polypeptide drugs, protein drugs and At least one of the genetic drugs.

Optionally, the dual-effect inhibitor of VEGFA signaling and FGF-2 signaling is used as a single active ingredient or together with other pharmaceutically acceptable active ingredients to form the drug.

Optionally, the FGF-2 signaling inhibitor is used as a single active ingredient or together with other pharmaceutically acceptable active ingredients to form the drug.

Optionally, the tumor includes a solid tumor, and the solid tumor includes liver cancer, lung cancer, pancreatic cancer, renal cancer, gastric cancer, esophageal cancer, colon cancer, bladder cancer, breast cancer, ovarian cancer, cervical cancer, clear cell renal cell carcinoma , skin melanoma, fibroma and nasopharyngeal carcinoma at least one.

Further, the solid tumor includes nasopharyngeal carcinoma.

Optionally, the tumor is a solid tumor expressing FGF-2.

In a second aspect, the present application provides the application of FGF-2 signaling inhibitors in the preparation of drugs for improving drug resistance of tumors to VEGFA signaling inhibitors, and the tumors are resistant to VEGFA signaling inhibitors.

Optionally, the FGF-2 signaling inhibitor inhibits at least one of the FGF-2 ligand, FGFR1 receptor and its downstream signaling pathway key molecule MYC, and the FGF-2 signaling inhibitor includes chemical drugs, polypeptides At least one of drugs, protein drugs and gene drugs.

Optionally, the VEGFA signaling inhibitor inhibits at least one of the VEGFA ligand, VEGFR2 receptor and its downstream signaling pathway key molecule MYC, and the VEGFA signaling inhibitor includes chemical drugs, polypeptide drugs, protein drugs and At least one of the genetic drugs.

Optionally, the FGF-2 signaling inhibitor is used as a single active ingredient or together with other pharmaceutically acceptable active ingredients to form the drug.

Optionally, the tumor includes a solid tumor, and the solid tumor includes liver cancer, lung cancer, pancreatic cancer, renal cancer, gastric cancer, esophageal cancer, colon cancer, bladder cancer, breast cancer, ovarian cancer, cervical cancer, clear cell renal cell carcinoma , skin melanoma, fibroma and nasopharyngeal carcinoma at least one.

Further, the solid tumor includes nasopharyngeal carcinoma.

Optionally, the tumor is a solid tumor expressing FGF-2.

In a third aspect, the present application provides a drug for treating tumors resistant to VEGFA signaling inhibitors, the drug including at least one of the dual-effect inhibitors of VEGFA signaling and FGF-2 signaling and FGF-2 signaling inhibitors A sort of.

Optionally, the dual-effect inhibitor of VEGFA signaling and FGF-2 signaling includes lenvatinib.

In a fourth aspect, the present application provides a drug for improving tumor resistance to VEGFA signaling inhibitors, and the drug includes FGF-2 signaling inhibitors.

The advantages of the embodiments of the present application will be partially clarified in the following description, and part of them will be obvious according to the description, or can be known through the implementation of the embodiments of the present application.

Description of drawings

Figure 1 is a comparison chart of the expression levels of pro-angiogenic factors in various tumors, where A is a comparison chart of the expression levels of pro-angiogenic factors in tumor RNA expression profiles, and B is a comparison chart of the relative expression levels of FGF-2 in tumor cell lines .

Figure 2 shows the experimental results of drug resistance of nasopharyngeal carcinoma tumor-bearing mice to VEGFA signaling inhibitors, where A is the tumor volume of nasopharyngeal carcinoma tumor-bearing mice transplanted with 5-8F nasopharyngeal carcinoma cells, and B is the transplanted knockdown Tumor volume of nasopharyngeal carcinoma tumor-bearing mice with 5-8F nasopharyngeal carcinoma cells expressing FGF-2 gene, C is the result of tumor blood vessel density, D is the result of tumor lung metastasis.

Figure 3 is the results of the effect of FGF-2 in fibroids and breast cancer, where A is the tumor volume of fibroid tumor-bearing mice, B is the result of tumor vessel density in fibroid tumor-bearing mice, and C is the fibroid tumor-bearing mice. The result of tumor lung metastasis in tumor-bearing mice, D is the tumor volume of breast cancer tumor-bearing mice, E is the result of tumor vessel density in breast cancer tumor-bearing mice, F is the result of tumor lung metastasis in breast cancer tumor-bearing mice.

Figure 4 is the effect diagram of dual-effect inhibitors of VEGFA signal and FGF-2 signal, where A is the tumor volume, B is the result of tumor blood vessel density, and C is the result of tumor lung metastasis.

Detailed ways

The following description is a preferred implementation of the embodiment of the present application. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the embodiment of the present application, some improvements and modifications can also be made. These improvements and retouching are also regarded as the scope of protection of the embodiments of the present application.

The present application provides the application of VEGFA signal and FGF-2 signal dual-effect inhibitor and/or FGF-2 signal inhibitor in the preparation of drugs for treating tumors, and tumors are drug-resistant to VEGFA signal inhibitors. That is to say, in one embodiment, the drug can include a dual-effect inhibitor of VEGFA signaling and FGF-2 signaling; in another embodiment, the drug can include an inhibitor of FGF-2 signaling; in yet another embodiment, the drug Dual inhibitors of VEGFA signaling and FGF-2 signaling may be included, as well as inhibitors of FGF-2 signaling.

In the embodiment of the present application, the dual-effect inhibitor of VEGFA signaling and FGF-2 signaling can simultaneously inhibit the VEGFA signaling pathway and FGF signaling pathway of endothelial cells in tumors, thereby overcoming the drug resistance of tumors to VEGFA signaling inhibitors and inhibiting tumor blood vessels. newborn. Dual inhibitors of VEGFA signaling and FGF-2 signaling inhibit the expression of VEGFA signaling, FGF-2 signaling, and downstream signaling molecules such as MYC, thereby inhibiting the proliferation of vascular endothelial cells, thereby hindering angiogenesis, inhibiting tumor cell proliferation, and reducing tumor volume , promote the apoptosis of tumor cells, and then achieve the effect of treating tumors, can effectively treat tumors resistant to VEGFA signal inhibitors, and optimize the therapeutic effect of existing tumors.

In this application, FGF-2 signaling inhibitors can effectively improve and treat (eliminate) the drug resistance of tumors to VEGFA signaling inhibitors. Specifically, FGF-2 signaling inhibitors inhibit the expression of downstream signaling molecules such as MYC through the FGF-2 receptor on tumor endothelial cells, thereby inhibiting the proliferation of vascular endothelial cells, thereby hindering angiogenesis, inhibiting tumor cell proliferation, reducing Tumor volume, promote tumor cell apoptosis, and then achieve the effect of tumor treatment.

In the embodiment of the present application, the dual-effect inhibitor of VEGFA signaling and FGF-2 signaling includes at least one of chemical drugs, polypeptide drugs, protein drugs and gene drugs that simultaneously inhibit VEGFA signaling and FGF-2 signaling pathways. In one embodiment, the chemical drug of the dual-effect inhibitor may be a small molecule organic compound. In another embodiment, the protein drugs of dual-effect inhibitors include but are not limited to natural proteins, recombinant proteins, and the like. In yet another embodiment, the gene-based drugs of dual-effect inhibitors include but are not limited to nucleic acid fragments, such as DNA fragments and RNA fragments. For example, the gene drug of the dual-effect inhibitor can be an siRNA drug that silences the expression of ligands and/or receptors and/or downstream signaling molecules in the VEGFA signaling pathway and the FGF-2 signaling pathway, and can also be a drug that down-regulates the target gene shRNA plasmids or shRNA lentiviral particles for protein expression.

In the embodiment of the present application, the dual-effect inhibitor of VEGFA signaling and FGF-2 signaling includes lenvatinib.

In the embodiment of the present application, the FGF-2 signaling inhibitor inhibits at least one of the FGF-2 ligand, FGFR1 receptor and its downstream signaling pathway key molecule MYC, and the FGF-2 signaling inhibitor includes chemical drugs and polypeptide drugs , at least one of protein drugs and gene drugs.

In one embodiment, the chemical drug of the FGF-2 signaling inhibitor can be a small molecule organic compound. For example, chemical drugs that inhibit FGF-2 signaling include AP24534, a FGFR1 tyrosine kinase inhibitor that inhibits FGF-2 signaling, and the like. In another embodiment, protein drugs for FGF-2 signaling inhibitors include but are not limited to natural proteins, recombinant proteins, and the like. Protein drugs such as FGF-2 signaling inhibitors include polypeptides that block the combination of FGF-2 and FGFR, such as FR-1039 and the like. In yet another embodiment, the genetic drug of FGF-2 signaling inhibitor includes but not limited to nucleic acid fragments, such as DNA fragments and RNA fragments. For example, the gene drug of FGF-2 signaling inhibitor can be an siRNA drug that silences the expression of ligands and/or receptors and/or downstream signaling molecules in the FGF-2 signaling pathway, and can also be a protein that down-regulates target genes Express shRNA plasmids or shRNA lentiviral particles.

In the embodiment of the present application, the VEGFA signal inhibitor inhibits at least one of VEGFA ligand, VEGFR2 receptor and its downstream signaling pathway key molecule MYC, and the VEGFA signal inhibitor includes chemical drugs, polypeptide drugs, protein drugs and gene drugs at least one of the drugs.

In the embodiment of the present application, the dual-effect inhibitor of VEGFA signaling and FGF-2 signaling is used as a single active ingredient or constitutes a drug together with other pharmaceutically acceptable active ingredients.

In the embodiments of the present application, the FGF-2 signaling inhibitor is used as a single active ingredient or constitutes a drug together with other pharmaceutically acceptable active ingredients. In one embodiment, other pharmaceutically acceptable active ingredients in the medicament include VEGFA signaling inhibitors.

In the embodiment of the present application, the tumor includes a solid tumor. In the embodiment of this application, solid tumors include liver cancer, lung cancer, pancreatic cancer, kidney cancer, gastric cancer, esophageal cancer, colon cancer, bladder cancer, breast cancer, ovarian cancer, cervical cancer, renal clear cell carcinoma, skin melanoma, fibrous At least one of tumor and nasopharyngeal carcinoma. In one embodiment, the solid tumor comprises nasopharyngeal carcinoma. Dual-effect inhibitors of VEGFA signaling and FGF-2 signaling can effectively treat nasopharyngeal carcinoma resistant to VEGFA signaling inhibitors, and FGF-2 signaling inhibitors can treat nasopharyngeal carcinoma resistant to VEGFA signaling inhibitors. The characteristic of nasopharyngeal carcinoma is that its tumor cells highly express FGF-2; FGF-2 can activate the expression of downstream signaling molecules such as MYC through one of its highly expressed receptors on endothelial cells, FGFR1, thereby alternatively activating endothelial cells Proliferation and angiogenesis, leading to the drug resistance effect of VEGFA signaling inhibitors unable to inhibit angiogenesis.

In the embodiments of the present application, the tumor is a solid tumor expressing FGF-2. Furthermore, solid tumors highly express FGF-2, which improves the targeting of drugs. In the embodiment of the present application, the tumor is a solid tumor expressing VEGFA. In the embodiment of the present application, the tumor is a solid tumor expressing FGF-2 and VEGFA.

In the embodiment of the present application, the medicine may also include pharmaceutically acceptable carriers and/or excipients. At this time, the drug simultaneously contains a dual-effect inhibitor of VEGFA signaling and FGF-2 signaling and/or an inhibitor of FGF-2 signaling, as well as pharmaceutically acceptable carriers and/or adjuvants. In this application, the function of "pharmaceutically acceptable carrier" is to transport the drug in this application so that it can play its due role. Generally speaking, transportation is from one organ or a certain part to another organ or another part. The carrier must be compatible with the drug ingredients, not affect the biological activity of the drug, and relatively non-toxic. It will not cause toxic side effects or serious reactions with the drugs it carries, and it will not have negative effects on patients.

In the embodiments of the present application, the pharmaceutically acceptable carrier includes at least one of solvent, polymer, liposome, recombinant viral vector and eukaryotic recombinant expression vector, but is not limited thereto. In one embodiment, the solvent includes but not limited to at least one of water, physiological saline, and other non-aqueous solvents. In another embodiment, the polymer comprises polylysine, polyethyleneimine (branched and/or chained) and its modifications, polyamidoamine dendrimers (PAMAM) and its derivatives , polypropyleneimine dendrimer (PPI) and its derivatives, chitosan, polylactic-glycolic acid (PLGA), polylactic acid, gelatin, cyclodextrin, sodium alginate, albumin and hemoglobin one or more, but not limited to. Among them, polyethyleneimine and its modified products, PAMAM and its derivatives, PPI and its derivatives, chitosan, etc. can be called cationic polymers. In another embodiment, liposomes can be self-assembled by cationic lipids, neutral auxiliary lipids, cholesterol, phospholipids (such as soybean lecithin, egg yolk lecithin, cephalin, etc.), and can also be formed by distearyl Acylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG) is interspersed in the phospholipid layer formed by phospholipid molecules. In another embodiment, the recombinant viral vector may include one or more of lentiviral vectors, adenoviral vectors and retroviral vectors, but is not limited thereto.

In this application, the active ingredients in the drug, VEGFA signal and FGF-2 signal dual-effect inhibitor and/or FGF-2 signal inhibitor can be dispersed or adsorbed in the above-mentioned carrier to form a dispersion system, or can be formed by the above-mentioned lipid body, polymer, etc., to form a spherical structure (such as nanocapsules or microcapsules). For example, when the dual-effect inhibitor of VEGFA signaling and FGF-2 signaling and/or the inhibitor of FGF-2 signaling can be a nucleic acid fragment, the drug can include, but not limited to, cationic polymers, polypeptides that encapsulate, bind or blend nucleic acid fragments , protein drugs, etc. Among them, the active ingredient encapsulated in the spherical structure can be released slowly, controlled or targeted, so that the drug can exert the best performance and improve the stability of the drug. For example, albumin, gelatin, chitosan, and polylactic acid can generally form microspheres capable of dispersing or encapsulating pharmaceutical active ingredients.

In the embodiment of the present application, the auxiliary material includes at least one of a diluent and an excipient. The main function of the diluent is to fill the weight or volume of the tablet to facilitate compression. In one embodiment, the diluent includes one or more of starches, sugars, celluloses and inorganic salts. Excipients refer to the additions in the drug other than the main drug active ingredients. In one embodiment, excipients include binders, fillers, disintegrants, lubricants in tablets, wine, vinegar, concoction, etc. in pills, bases in semi-solid ointments and creams Part, preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solubilizers, osmotic pressure regulators, colorants, etc. in liquid preparations.

In the embodiment of the present application, the form of the drug includes tablet, capsule, powder, granule, pill, syrup, solution or suspension. The specific application form depends on the actual situation. In the embodiment of the present application, the drug can be administered orally or by injection. When administered by injection, the pharmaceutical form is preferably a solution, for example dissolved in water or physiological saline. In one embodiment, the injection can be administered by intraperitoneal injection, subcutaneous injection, intramuscular injection or intravenous injection. In the embodiment of the present application, the drug can be administered locally or systemically.

In the embodiment of the present application, the dosage of the drug is 0.1-100 mg/kg body weight per day. Specifically, the dosage of the drug depends on various factors, including but not limited to the desired biological activity and the tolerance of the drug to the subject.

Nasopharyngeal carcinoma naturally has high expression of FGF-2. In nasopharyngeal carcinoma or solid tumors with high FGF-2 expression, a series of molecular biology experiments have confirmed that FGF-2 signaling can act on FGFR1 receptors on endothelial cells in tumors. body, activate downstream signaling pathways such as MYC, etc., and promote the proliferation of vascular endothelial cells. The classic VEGFA signal can act on the VEGFR2 receptor on the endothelial cells in the tumor, activate downstream signaling pathways such as MYC, etc., and promote the proliferation of vascular endothelial cells. Since the two signaling pathways share the same downstream signaling molecule, FGF-2 can compensatory maintain angiogenesis in the event of VEGFA signaling inhibition, resulting in tumor resistance to VEGFA signaling inhibitors. Dual-effect inhibitors of VEGFA signaling and FGF-2 signaling can simultaneously inhibit these two signals, breaking the drug resistance mechanism of tumors, thereby effectively inhibiting tumor angiogenesis and improving the curative effect on tumors. The new use of the VEGFA signal and FGF-2 signal dual-effect inhibitor provided by the application opens up a new approach for the treatment of nasopharyngeal carcinoma or solid tumors with high FGF-2 expression, and has a more significant therapeutic effect.

The present application also provides the application of the FGF-2 signal inhibitor in the preparation of drugs for improving the drug resistance of tumors to VEGFA signal inhibitors, and the tumors have drug resistance to VEGFA signal inhibitors.

In this application, the application of FGF-2 signaling inhibitors can effectively improve and treat (eliminate) the drug resistance of tumors to VEGFA signaling inhibitors, thereby optimizing the tumor therapeutic effect of existing VEGFA signaling inhibitors. Specifically, FGF-2 signaling inhibitors inhibit the expression of downstream signaling molecules such as MYC through the FGF-2 receptor on tumor endothelial cells, thereby inhibiting the proliferation of vascular endothelial cells, thereby hindering angiogenesis, inhibiting tumor cell proliferation, reducing Tumor volume, promote tumor cell apoptosis, and then achieve the effect of tumor treatment.

In the embodiment of the present application, the FGF-2 signaling inhibitor inhibits at least one of the FGF-2 ligand, FGFR1 receptor and its downstream signaling pathway key molecule MYC, and the FGF-2 signaling inhibitor includes chemical drugs and polypeptide drugs , at least one of protein drugs and gene drugs. Specific types of FGF-2 signaling inhibitors have been described above and will not be repeated here.

In the embodiment of the present application, the VEGFA signal inhibitor inhibits at least one of VEGFA ligand, VEGFR2 receptor and its downstream signaling pathway key molecule MYC, and the VEGFA signal inhibitor includes chemical drugs, polypeptide drugs, protein drugs and gene drugs at least one of the drugs.

In the embodiments of the present application, the FGF-2 signaling inhibitor is used as a single active ingredient or constitutes a drug together with other pharmaceutically acceptable active ingredients.

In the embodiment of the present application, the tumor includes a solid tumor. In the embodiment of this application, solid tumors include liver cancer, lung cancer, pancreatic cancer, kidney cancer, gastric cancer, esophageal cancer, colon cancer, bladder cancer, breast cancer, ovarian cancer, cervical cancer, renal clear cell carcinoma, skin melanoma, fibrous At least one of tumor and nasopharyngeal carcinoma. In one embodiment, the solid tumor comprises nasopharyngeal carcinoma. FGF-2 signaling inhibitors can effectively treat nasopharyngeal carcinoma resistant to VEGFA signaling inhibitors, and FGF-2 signaling inhibitors can treat nasopharyngeal carcinoma resistant to VEGFA signaling inhibitors. The characteristic of nasopharyngeal carcinoma is that its tumor cells highly express FGF-2; FGF-2 can activate the expression of downstream signaling molecules such as MYC through one of its highly expressed receptors on endothelial cells, FGFR1, thereby alternatively activating endothelial cells Proliferation and angiogenesis, leading to the drug resistance effect of VEGFA signaling inhibitors unable to inhibit angiogenesis.

In the embodiments of the present application, the tumor is a solid tumor expressing FGF-2. Furthermore, solid tumors highly express FGF-2, which improves the targeting of drugs. In the embodiment of the present application, the tumor is a solid tumor expressing VEGFA. In the embodiment of the present application, the tumor is a solid tumor expressing FGF-2 and VEGFA.

In the embodiment of the present application, the medicine may also include pharmaceutically acceptable carriers and/or excipients. The selection of carriers and excipients in the drug for improving tumor resistance to VEGFA signaling inhibitors is as described above; the form, administration method, and dosage of the drug are described in the above-mentioned drugs for the treatment of tumors, and will not be repeated here.

Nasopharyngeal carcinoma naturally has a high expression of FGF-2. In nasopharyngeal carcinoma or solid tumors with high FGF-2 expression, FGF-2 signaling can act on the FGFR1 receptor on endothelial cells in the tumor, activate downstream signaling pathways, and promote vascular Endothelial cells proliferate and maintain angiogenesis compensatoryly, resulting in resistance to inhibitors of VEGFA signaling. The use of FGF-2 signaling inhibitors can block this drug resistance mechanism of tumors, thereby effectively reversing the drug resistance of tumors to VEGFA signaling inhibitors, and improving the effect of VEGFA signaling inhibitors on nasopharyngeal carcinoma or entities with high FGF-2 expression. Tumor efficacy. The new use of the FGF-2 signaling inhibitor provided by this application can effectively improve the drug resistance of nasopharyngeal carcinoma and other tumors to VEGFA signaling inhibitors, and optimize the therapeutic effect of the existing VEGFA signaling inhibitors.

The present application also provides a drug for treating tumors resistant to VEGFA signal inhibitors. The drug includes at least one of a dual-effect inhibitor of VEGFA signal and FGF-2 signal and an FGF-2 signal inhibitor.

In the embodiment of the present application, the dual-effect inhibitor of VEGFA signaling and FGF-2 signaling includes lenvatinib.

In one embodiment, the drug for treating tumors resistant to VEGFA signaling inhibitors includes dual inhibitors of VEGFA signaling and FGF-2 signaling. In another embodiment, the drug for treating tumors resistant to VEGFA signaling inhibitors includes FGF-2 signaling inhibitors and VEGFA signaling inhibitors.

In this application, the medicine may also include carriers and/or excipients; the medicine may also include other pharmaceutically acceptable active ingredients. The selection of carriers and auxiliary materials is as described above, and will not be repeated here.

The present application also provides a drug for improving tumor resistance to VEGFA signal inhibitors, and the drug includes FGF-2 signal inhibitors.

In this application, the medicine may also include carriers and/or excipients; the medicine may also include other pharmaceutically acceptable active ingredients. The selection of carriers and auxiliary materials is as described above, and will not be repeated here.

Introduction to the experiment in this application:

(1) Cell culture

The human nasopharyngeal carcinoma cell lines 5-8F, HONE-1, and CNE1 used in this application were kindly provided by Shenzhen Second People's Hospital. The human A549 lung cancer cell line was kindly provided by the Department of Cellular and Genetic Medicine, Fudan University. Human SK-MEL-5 melanoma, HepG2 hepatocellular carcinoma, MDA-MB-231 breast cancer, SW480 colon cancer, PANC-1 pancreatic ductal adenocarcinoma, 293T embryonic kidney cells, mouse T241 fibroma cell line and FGF-2 The expressed T241 fibroma cell line, mouse 4T1 breast cancer cell line and FGF-2 overexpressed 4T1 breast cancer cell line were kindly provided by Karolinska Institutet, Sweden. Cells were placed in 10% FBS (catalog number 40130ES76, YEASEN) DMEM medium (catalogue number MA0213, Meilunbio) or RPMI 1640 medium containing 100 U/mL penicillin, 100 μg/mL streptomycin (catalogue number MA0110, Meilunbio) (Cat. No. MA0215, Meilunbio) for cultivation. In the FGF-2 gene shRNA experiment, nasopharyngeal carcinoma tumor cells were stably transfected with lentiviral vector produced by 293T embryonic kidney cells.

(2) Animal experiments

The animal experiments in this application used male BALB/c-nu/nu nude mice, male C57/B6 mice, or female BALB/c mice aged 6-8 weeks, which were purchased from Jizui Yaokang Biotechnology Company. Mice were maintained under standard animal housing conditions and fed a standard diet. When constructing mouse xenograft tumor models and mouse xenograft tumor models, 1×10 ⁶ 5-8F nasopharyngeal carcinoma cells, T241 fibroma cells or 4T1 breast cancer cells suspended in 100 μL PBS were implanted into nude mice Subcutaneously, subcutaneously in C57/B6 mice, or in the mammary fat pad of BALB/c mice, tumor size was measured with calipers every other day, and the transplanted tumor volume was calculated according to standard formulas.

When investigating the therapeutic effects of different pharmaceutical preparations on mice of different tumor models, the pharmaceutical preparations can be intragastrically or intraperitoneally injected into mice of different experimental groups. After a period of time, the mice were sacrificed, dissected and subjected to histological examination, RNA and protein extraction, tumor blood vessel density and other examinations.

When discussing tumor metastasis, when the tumor size is 2.0cm ³ , resection of the primary tumor in mice was performed. After the mice were anesthetized with an air anesthesia machine, the tumors were surgically removed. After the completion, feed the water containing antibiotics, pay attention to the recovery of the body temperature of the mice, and observe closely. After the mice recover their normal activity, place them in a cage and raise them separately. Check the wound healing and the activity of the mice every 4-6 hours, and continue after the wounds are completely healed. feeding. 4-6 weeks after the subcutaneous tumor resection, the mice were sacrificed, dissected and subjected to histological examination, lung metastasis examination, histological examination, etc.

Experiment 1: VEGFA signaling inhibitor-resistant nasopharyngeal carcinoma specifically overexpresses FGF-2 compared with VEGFA signaling inhibitor-resistant colon cancer

Results of clinical trials have found that colon cancer is less resistant to VEGFA signaling inhibitors. VEGFA signaling inhibitors such as bevacizumab have become an important first-line therapy for colon cancer. However, drug resistance to VEGFA signaling inhibitors often occurs in nasopharyngeal carcinoma, so bevacizumab does not perform well in clinical trials for the treatment of nasopharyngeal carcinoma. Figure 1 is a comparison chart of the expression levels of pro-angiogenic factors in various tumors, where A is a comparison chart of the expression levels of pro-angiogenic factors in tumor RNA expression profiles, and B is a comparison chart of the relative expression levels of FGF-2 in tumor cell lines . In Figure 1 A, the large-scale human tumors of various tumor types in the TCGA public database and the Oncomine database (clear cell renal cell carcinoma KIRC/colon cancer COAD/nasopharyngeal carcinoma NPC/gastric cancer STAD/pancreatic cancer PAAD/lung adenocarcinoma LUAD/breast cancer BRCA/skin melanoma SKCM/) RNA expression profile results were compared and analyzed across platforms. Fibroblast growth factor 2 (FGF-2/ platelet-derived growth factor B PDGFB/ epidermal growth factor EGF/ angiopoietin 1 ANGPT1/ erythropoietin EPO), FGF-2 is specifically highly expressed in nasopharyngeal carcinoma, while in Lowest expression in colon cancer. In Figure 1B, using various types of human tumor cell lines, it was found that three nasopharyngeal carcinoma (NPC) cell lines all expressed FGF-2 at high levels, while human colon cancer cells (SW480) expressed FGF-2 levels at The lowest in the tested group. Therefore, Figure 1 shows that FGF-2 is expressed at high levels in NPC.

Experiment 2: Nasopharyngeal carcinoma tumor-bearing mice are resistant to VEGFA signaling inhibitors, and knocking down the FGF-2 gene in nasopharyngeal carcinoma cells can improve the resistance of nasopharyngeal carcinoma tumor-bearing mice to VEGFA signaling inhibitors

5-8F nasopharyngeal carcinoma tumor-bearing mice were constructed. When the tumor grew to 0.5cm ³ , the mice were randomly divided into two groups. One group was the experimental group, which was treated with VEGFA signal inhibitor (bevacizumab) (Anti -VEGF), and the other group was the control group, which was treated with PBS buffer (Vehicle). Each group was intraperitoneally injected with a concentration of 2.5mg/kg, twice a week. Figure 2 shows the experimental results of drug resistance of nasopharyngeal carcinoma tumor-bearing mice to VEGFA signaling inhibitors, where A is the tumor volume of nasopharyngeal carcinoma tumor-bearing mice transplanted with 5-8F nasopharyngeal carcinoma cells, and B is the transplanted knockdown Tumor volume of nasopharyngeal carcinoma tumor-bearing mice with FGF-2 gene 5-8F nasopharyngeal carcinoma cells, C is the result of tumor blood vessel density, D is the result of tumor lung metastasis.

Figure 2A shows that nasopharyngeal carcinoma tumor-bearing mice transplanted with 5-8F nasopharyngeal carcinoma cells (NPC-shScrambled) are resistant to VEGFA signaling inhibitors, and there is no significant difference in tumor volume between the experimental group and the control group, that is, the two There was no difference in tumor growth between groups. In Figure 2B, the same experiment as above was carried out using 5-8F nasopharyngeal carcinoma cells knocked down the FGF-2 gene. Tumor-bearing mice (NPC-shFGF2) were not resistant to VEGFA signaling inhibitors, and tumor growth was significantly inhibited by VEGFA signaling inhibitors.

Further, it can be seen from Fig. 2C that in the nasopharyngeal carcinoma tumors of the control group, the blood vessel density was not affected by the VEGFA signaling inhibitor. However, in nasopharyngeal carcinoma tumors knocked down the FGF-2 gene, the blood vessel density was significantly inhibited when treated with the same drug and the same conditions.

Furthermore, in D of Figure 2, the same tumor-bearing mice as above were used for tumor metastasis experiments, and it was found that in the control group nasopharyngeal carcinoma tumors, the incidence of lung metastasis was not significantly affected by VEGFA signaling inhibitors; In nasopharyngeal carcinoma tumors with low FGF-2 gene, the incidence of lung metastasis was significantly suppressed by using the same drug and treatment under the same conditions. Therefore, Figure 2 demonstrates that blocking FGF-2 signaling can improve the resistance of NPC to VEGFA signaling inhibitors.

Experiment 3: Other types of tumors with high expression of FGF-2 can increase tumor growth, blood vessel density and metastasis

Tumor-bearing mice with T241 fibroids in the control group and T241 fibroids with high expression of FGF-2 were constructed, which were T241-Vector group and T241-FGF-2 group, respectively. The tumor-bearing mice of control group 4T1 breast cancer and 4T1 breast cancer with high expression of FGF-2 were constructed as 4T1-Vector group and 4T1-FGF-2 group, respectively. The effect of FGF-2 in fibroids and breast cancer was studied, and the results are shown in Figure 3, where A is the tumor volume of fibroid tumor-bearing mice, B is the result of tumor blood vessel density of fibroid tumor-bearing mice, and C is the result of tumor lung metastasis in fibroid tumor-bearing mice, D is the tumor volume of breast cancer tumor-bearing mice, E is the result of tumor vessel density in breast cancer tumor-bearing mice, F is the tumor lung in breast cancer tumor-bearing mice transfer results.

It can be seen from A-C of Figure 3 that using T241 fibroma cells with high expression of FGF-2 gene, the tumor growth is significantly enhanced, and the blood vessel density is significantly increased; the tumor metastasis experiment using tumor-bearing mice shows that the incidence of lung metastasis is significantly enhanced . From D-F of Figure 3, it can be seen that using 4T1 breast cancer cells with high expression of FGF-2 gene, the tumor growth was significantly enhanced, and the blood vessel density was significantly increased; the tumor metastasis experiment was performed on tumor-bearing mice, and the incidence of lung metastasis was significantly increased. enhanced. Figure 3 shows that in other tumors other than nasopharyngeal carcinoma, high expression of FGF-2 signaling can enhance angiogenesis, thus indicating that the mechanism of experiment 1 can also occur in other tumor types with high expression of FGF-2 signaling.

Experiment 4: Dual inhibitors of VEGFA signaling and FGF-2 signaling can treat nasopharyngeal carcinoma tumor-bearing mice resistant to VEGFA signaling inhibitors

5-8F nasopharyngeal carcinoma tumor-bearing mice were constructed, and when the tumor grew to 0.5cm ³ , the mice were randomly divided into three groups, one group was the experimental group, and the double-effect inhibitor of VEGFA signal and FGF-2 signal (Lenval Bevacizumab) was treated (Lenvatinib); one group was the control group, which was treated with a VEGFA signaling inhibitor (bevacizumab) (Anti-VEGF); one group was the blank group, which was treated with a solvent (Vehicle). Among them, bevacizumab was intraperitoneally injected at a concentration of 2.5 mg/kg, twice a week; lenvatinib was administered intragastrically at a concentration of 30 mg/kg, once a day (preliminary experiments showed that bevacizumab and lenvatinib at the above concentrations The effect of Tini on non-drug-resistant colon cancer is the same), and the effect of dual-effect inhibitors of VEGFA signaling and FGF-2 signaling was studied. The results are shown in Figure 4, where A is the tumor volume, B is the result of tumor blood vessel density, C is the result of tumor lung metastasis.

In Figure 4A, it can be seen that nasopharyngeal carcinoma tumor-bearing mice have inconsistent responses to VEGFA signaling and FGF-2 signaling dual-effect inhibitors and VEGFA signaling inhibitors, and their responses to VEGFA signaling inhibitors (bevacizumab) Resistant to VEGFA signaling and FGF-2 signaling dual inhibitors (lenvatinib), indicating that VEGFA signaling and FGF-2 signaling dual inhibitors are effective in treating resistance to VEGFA signaling inhibitors of nasopharyngeal carcinoma.

Furthermore, it can be seen in B of Figure 4 that the vessel density in nasopharyngeal carcinoma tumors was not affected by the VEGFA signaling inhibitor; while in the VEGFA signal and FGF-2 signal dual-effect inhibitor treatment group, the vessel density was significantly inhibited.

Furthermore, in Figure 4C, the same tumor-bearing mice were used to carry out tumor metastasis experiments, and it was found that in nasopharyngeal carcinoma tumors, the incidence of lung metastasis was not significantly affected by VEGFA signaling inhibitors; while VEGFA signaling and FGF- In the 2-signal dual-effect inhibitor treatment group, the incidence of lung metastasis was significantly suppressed. Therefore, Experiment 4 shows that the use of dual inhibitors of VEGFA signaling and FGF-2 signaling can effectively treat nasopharyngeal carcinoma.

The above-mentioned embodiments only express several implementation modes of the present application, and the description thereof is relatively specific and detailed, but should not be construed as limiting the patent scope of the present application. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the scope of protection of the patent application should be based on the appended claims.

Claims (10)

1. Use of a VEGFA signalling and a dual effect inhibitor of FGF-2 signalling and/or an inhibitor of FGF-2 signalling for the preparation of a medicament for the treatment of tumours which are resistant to VEGFA signalling inhibitors.
2. 2. The use of claim 1, wherein said dual-effect inhibitor of VEGFA signaling and FGF-2 signaling is capable of inhibiting both the VEGFA signaling pathway and the FGF-2 signaling pathway of endothelial cells in said tumor, thereby overcoming resistance of said tumor to said inhibitor of VEGFA signaling and inhibiting tumor angiogenesis.
3. 3. The use of claim 1, wherein the dual-effect inhibitor of VEGFA signaling and FGF-2 signaling comprises ranvatinib;

   the FGF-2 signal inhibitor inhibits at least one of FGF-2 ligand, FGFR1 receptor and downstream signaling pathway key molecule MYC, and the FGF-2 signal inhibitor comprises at least one of chemical drugs, polypeptide drugs, protein drugs and gene drugs;

   the VEGFA signal inhibitor inhibits at least one of VEGFA ligand, VEGFR2 receptor and downstream signal pathway key molecule MYC, and comprises at least one of chemical drugs, polypeptide drugs, protein drugs and gene drugs;

   the VEGFA signal and FGF-2 signal double-effect inhibitor is used as a single active ingredient or forms the medicament with other pharmaceutically acceptable active ingredients;

   the FGF-2 signaling inhibitor is used as a single active ingredient or forms the medicament together with other pharmaceutically acceptable active ingredients.
4. 4. The use of claim 1, wherein the tumor comprises a solid tumor comprising at least one of liver cancer, lung cancer, pancreatic cancer, kidney cancer, stomach cancer, esophageal cancer, colon cancer, bladder cancer, breast cancer, ovarian cancer, cervical cancer, renal clear cell carcinoma, skin melanoma, fibroids, and nasopharyngeal cancer; further, the solid tumor includes nasopharyngeal carcinoma;

   the tumor is a solid tumor expressing FGF-2.
5. Use of an FGF-2 signalling inhibitor for the preparation of a medicament for improving the resistance of a tumour to a VEGFA signalling inhibitor, said tumour being resistant to a VEGFA signalling inhibitor.
6. 6. The use of claim 5, wherein the FGF-2 signaling inhibitor inhibits at least one of an FGF-2 ligand, an FGFR1 receptor, and a downstream signaling pathway key molecule MYC thereof, and wherein the FGF-2 signaling inhibitor comprises at least one of a chemical drug, a polypeptide drug, a protein drug, and a gene drug;

   the VEGFA signal inhibitor inhibits at least one of VEGFA ligand, VEGFR2 receptor and downstream signal pathway key molecule MYC, and comprises at least one of chemical drugs, polypeptide drugs, protein drugs and gene drugs;

   the FGF-2 signaling inhibitor is used as a single active ingredient or forms the medicament together with other pharmaceutically acceptable active ingredients.
7. 7. The use of claim 5, wherein the tumor comprises a solid tumor comprising at least one of liver cancer, lung cancer, pancreatic cancer, kidney cancer, stomach cancer, esophageal cancer, colon cancer, bladder cancer, breast cancer, ovarian cancer, cervical cancer, renal clear cell carcinoma, skin melanoma, fibroids, and nasopharyngeal cancer; further, the solid tumor includes nasopharyngeal carcinoma;

   the tumor is a solid tumor expressing FGF-2.
8. 8. A medicament for treating a tumor that is resistant to a VEGFA signaling inhibitor, comprising VEGFA signaling and at least one of a dual-effect inhibitor of FGF-2 signaling and an inhibitor of FGF-2 signaling.
9. 9. The medicament of claim 8, wherein the dual-effect inhibitor of VEGFA signaling and FGF-2 signaling comprises ranvatinib.
10. 10. A medicament for improving the resistance of a tumour to VEGFA signalling inhibitors, characterised in that the medicament comprises an FGF-2 signalling inhibitor.

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